Project Summary/Abstract The goal of this revised R01 is to determine how a hypothalamic region integrates and then processes hormone and CNS metabolic cues to affect sex-dependent physical activity behavior in female mice. We showed previously that loss of hormone-responsive neurons in the medial basal hypothalamus (MBH) diminishes activity only in female mice. In new work, we discovered that a small, discrete cluster of melanocortin 4 receptor (Mc4r)-expressing neurons in the ventrolateral region of the ventromedial hypothalamus (VMHvlMC4R neurons) increases female activity and promotes weight loss when activated. Increasing dosage of Mc4r in the VMHvl of wild type female mice by CRISPRa increases distance traveled leading to weight loss when pair-fed with control mice. Here, we will ask if this VMHvlMC4R node can be exploited to mitigate age-related and environmental- induced metabolic challenges and also define the mechanistic pathway and circuits responsible for regulating this module. Aim 1 will ask if the VMHvlMC4R node depends on hormones, can improve metabolic deficits, and whether silencing this node (inhibitory DREADDs) results in lowered physical activity behaviors. Aim 2 will confirm that Mc4r is an integral part of this node and define other signaling components that participate in the VMHvlMC4R activity node. Aim 3 will address the physiological consequences of melanocortin and estrogen signaling in modulating VMHvlMC4R neuron activity by electrophysiology brain slice recordings with the Gao lab (Yale) and begin mapping inputs that interact with VMHvlMC4R neurons. We posit that this ancillary VMHvlMC4R female-module disengages in states of low or no estrogen (estrus or post-menopause period) thereby reducing metabolic fitness. Our approaches to define the molecular basis of this VMHvl activity node are highly innovative and likely to provide novel insights into how fluctuating hormone-status in a normal cycling female or during different life-stages drives physical activity behaviors.